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. 2018 Aug 17:9:1883.
doi: 10.3389/fmicb.2018.01883. eCollection 2018.

High-Throughput Identification of Candidate Strains for Biopreservation by Using Bioluminescent Listeria monocytogenes

Sara M El Kheir  1   2 , Lamia Cherrat  1 , Ahoefa A Awussi  1 , Nancy E Ramia  1   2 , Samir Taha  2 , Abdur Rahman  3 , Delphine Passerini  4 , Françoise Leroi  4 , Jeremy Petit  1 , Cécile Mangavel  1 , Anne-Marie Revol-Junelles  1 , Frédéric Borges  1
Affiliations

High-Throughput Identification of Candidate Strains for Biopreservation by Using Bioluminescent Listeria monocytogenes

Sara M El Kheir et al. Front Microbiol. .

Abstract

This article describes a method for high-throughput competition assays using a bioluminescent strain of L. monocytogenes. This method is based on the use of the luminescent indicator strain L. monocytogenes EGDelux. The luminescence of this strain is correlated to growth, which make it suitable to monitor the growth of L. monocytogenes in mixed cultures. To this aim, luminescence kinetics were converted into a single numerical value, called the Luminescence Disturbance Indicator (LDI), which takes into account growth inhibition phenomena resulting in latency increase, decrease in the luminescence rate, or reduction of the maximum luminescence. The LDI allows to automatically and simultaneously handle multiple competition assays which are required for high-throughput screening (HTS) approaches. The method was applied to screen a collection of 1810 strains isolated from raw cow's milk in order to identify non-acidifying strains with anti-L. monocytogenes bioprotection properties. This method was also successfully used to identify anti-L. monocytogenes candidates within a collection of Lactococcus piscium, a species where antagonism was previously described as non-diffusible and requiring cell-to-cell contact. In conclusion, bioluminescent L. monocytogenes can be used in HTS to identify strains with anti-L. monocytogenes bioprotection properties, irrespectively of the inhibition mechanism.

Keywords: Listeria monocytogenes; antibacterial activities; bioluminescence; biopreservation; bioprotection; competition; high-throughput screening assays; mixed culture.

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Figures

FIGURE 1
FIGURE 1
Growth kinetics and luminescence of L. monocytogenes EGDelux. (A) OD595 nm and luminescence. (B) Growth rate and luminescence. Error bars represent standard errors.
FIGURE 2
FIGURE 2
Impact of NaCl concentration on growth and luminescence of L. monocytogenes EGDelux. (A) biomass production kinetics, (B) luminescence kinetics, and (C) evolution of LDI with NaCl concentration. Error bars represent standard errors.
FIGURE 3
FIGURE 3
Impact of pH on growth and luminescence of L. monocytogenes EGDelux. (A,D) biomass production kinetics, (B,E) luminescence kinetics, and (C,F) evolution of LDI with pH. (A–C) acidic pH values and (D–F) basic pH values. Error bars represent standard errors.
FIGURE 4
FIGURE 4
Impact of nisin concentration on growth and luminescence by L. monocytogenes EGDelux. (A) biomass production kinetics, (B) luminescence kinetics, and (C) evolution of LDI with nisin concentration. Error bars represent standard errors.
FIGURE 5
FIGURE 5
Luminescence kinetics under different scenarii of growth inhibition. Solid line: control; dotted line: sample exhibiting luminescence changes owing to growth inhibition. Growth inhibition can lead to a reduced pic size of luminescence (A), a delayed pic (B), or a pic with decreased slopes (C). In these theoretical scenarii, the LDIr is of 50%.
See this image and copyright information in PMC

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